Human immunodeficiency virus (HIV) infection (which results in Acquired Immune Deficiency Syndrome, AIDS) is a relatively new infection in the human population, but it has quickly risen to one of the foremost health problems in the world. HIV/AIDS has now become the leading cause of death in sub-Saharan Africa, as well as the fourth biggest killer worldwide (BMJ 2001; 323(7324):1271). At the end of 2010, it was estimated that more than 34 million people were living with HIV infection worldwide, including around 16.8 million women and 3.4 million children (World Health Organisation data). In 2010, there were 2.7 million people newly infected with HIV, and the death toll due to AIDS reached 1.5 million people.
Low- and middle-income countries are the most plagued by HIV (about 97% of new infections are registered there), but adult and child deaths due to AIDS in 2010 were 30,000 in western industrialised countries.
Better treatment methods are now known to prolong the life of patients with HIV infection, but no cure has been found yet for this disease.
Current anti-HIV drugs target several different stages of the HIV life cycle, and several of the enzymes that HIV requires to replicate and survive (Arts et al. Cold Spring harb Perspect Med 2012:2(4):a007161). Some of the commonly used anti-HIV drugs include nucleoside/nucleotide reverse transcriptase inhibitors, NRTIs (such as emtricitabine, stavudine, ddl, ddC, d4T, 3TC, zidovudine, abacavir, tenofovir etc); non-nucleoside reverse transcriptase inhibitors, NNRTIs (such as rilpivirine, etravirine, nevirapine, efavirenz and delavirdine); protease inhibitors, PIs (such as saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, lopinavir and atazanavir); entry inhibitors, including fusion inhibitors (such as enfuvirtide, maraviroc, ibalizumab etc) and others, such as the integrase inhibitor isentress (raltegravir) (http://www.fda.gov/ForConsumers/byAudience/ForPatientAdvocates/HIVandAIDSActivities/ucm118915.htm).
The condition where T cells engage in uncontrolled cell division is called T cell hyperproliferation, leading to immune hyperactivation. T cells are immune system cells that can develop the capacity to kill infected or neoplastic cells. When T cells are contacted by antigens they become activated, or sensitised, and proliferate, that is, appear in greater numbers (Grossman et al. Nat Med 2006; 12; 289-295, Brenchley et al. Nat Med 2006; 12:1365-1371). This is a normal physiological process, which is useful to protect the host from “sick cells” (tumor cells and infected cells).
However, excessive T cell activation, and particularly prolonged, excessive activation can contribute to disease progression and is considered a key pathogenetic factor in several chronic diseases such as cancer and chronic infectious diseases including HIV/AIDS (Sodora et al. AIDS 2008 22:439-446, Hellerstein et al. J Clin Invest 2003; 112:956-966, Liovat et al. PLOS ONE 2012; 7(10):e46143, Cossarizza et al. PLOS ONE 2012:7(12):e50728, Hunt et al. AIDS 2011; 25(17):2123-2131).
In addition, HIV infection of T cells depends on active division and proliferation of such cells. Firstly, infected dividing T cells produce a large amount of HIV particles (approximately 8-10 fold more than in quiescent T cells). Secondly, antigenic stimulation by such HIV particles sustains further T cell activation or proliferation, as mentioned before. This results in a dangerous “vicious cycle”. In addition to direct antigenic stimulation by HIV, microbial translocation across impaired gut-associated lymphoid tissues (GALT) throughout the course of HIV disease also sustains elevated T cell activation/proliferation (Brenchley et al. Nat Med, 2006. 12(12): p. 1365-71). This chronic cycle of events, over time, exhausts the immune system. Therefore, limiting T cell hyperactivation and hyperproliferation will have a dual effect: it will not only suppress HIV replication, but it will also prevent the loss of functional CD4 T helper cells and slow down disease progression.
In order to try to address this unmet medical need we have tried to develop compounds designed to provide both an antiviral and an antiproliferative component. In order to establish the proof of concept for this approach in humans, we combined into a single capsule, two readily available generic drugs, hydroxyurea (HU) and didanosine (ddl), which we called VS411. In VS411, ddl is the antiviral component and HU is the antiproliferative component. The NRTI, ddl has been used extensively in the treatment of HIV in combination cocktails as a direct acting antiretroviral agent targeting the HIV encoded reverse transcriptase enzyme activity. HU is an antiproliferative agent indicated for the treatment of different neoplastic as well as non-neoplastic diseases such as sickle cell anemia and psoriasis. HU has been used for the treatment of HIV-infected individuals, especially in combination with antiretroviral drugs, such as ddl. HU inhibits the cellular enzyme ribonucleotide reductase, blocking the transformation of ribonucleotides into deoxyribonucleotides, thus depleting the intracellular deoxynucleotide triphosphate (dNTP) pool, and arresting the cell cycle in the G1/S phase (Lori AIDS, 1999; 13(12):1433-42). By depleting the dNTP pool, HU also strongly inhibits viral deoxyribonucleic acid (DNA) synthesis through the virally encoded reverse transcriptase. Moreover, HU can also suppress virus replication by slowing down the rate of T-cell proliferation (as stated above, HIV-1 needs actively dividing cells to optimally replicate).
VS411, was first studied in a Phase I clinical trial in which it exhibited a favourable safety profile and the best formulation was identified (De Forni et al. Br J Pharmacol 2010; 161(4):830-843). VS411 was then investigated in a Proof-of-Concept multinational Phase II trial. In the course of this Phase II study, VS411 exhibited excellent safety and tolerability profiles and reduction in viral load and immune activation after just 28 days of therapy. These results provided solid Proof-of-Concept evidence that therapeutics with antiviral and antiproliferative activities can provide a clinically significant benefit in the treatment of HIV/AIDS (Lori et al. PloS ONE 2012; 7(10): e47485. doi:10.1371/journal.pone.0047485).
Recently, attention has also been turned to cyclin-dependent kinases (CDKs), key regulators of the cell cycle and RNA polymerase II transcription. Cyclin-dependent kinases (CDKs) are non-receptor serine-threonine protein kinases that require cyclin for their activity and play a fundamental role in controlling cell cycle progression. Cell division is a highly regulated process responding to cellular signals both within the cell and from external sources (Gerard et al. Front Physiol 2012; 3:413).
To date, thirteen CDKs have been identified in humans (Chen et al. Biochem Biophys Res Commun. 2007, 354, 735-40; Mani et al. Exp. Opin. Invest. Drugs 2000; 9(8):1849-1870, Sergere et al. Biochem. Biophys. Res. Commun. 2000, 276, 271-277, Hu et al. J. Biochem. Chem. 2003; 278(1 0):8623-8629).
Because CDKs play an important role in the regulation of cellular proliferation, CDK inhibitors could be useful in the treatment of cell proliferative disorders such as cancer, neuro-fibromatosis, psoriasis, fungal infections, endotoxic shock, transplantation rejection, vascular smooth cell proliferation associated with artheroscelerosis, pulmonary fibrosis, arthritis, glomerulonephritis and post-surgical stenosis and restenosis (U.S. Pat. No. 6,114,365).
CDKs are also known to play a role in apoptosis. Therefore CDK inhibitors could be useful in the treatment of cancer; autoimmune diseases, for example systemic lupus, erythematosus, autoimmune mediated glomerulonephritis, rheumatoid arthritis, psoriasis, inflammatory bowel disease, and autoimmune diabetes (Roberts et al. J Natl Cancer 2012; 104(6):476-487, Zoja Arthritis Rheum 2007; 56:1629-1637). Indeed several pharmacological CDK inhibitors (PCIs) are currently in clinical trials as potential cancer therapeutics (Benson et al. Br J Cancer 2007; 96(1):29-37, Luke et al. Clin Cancer 2012; 18(9):2638-2647).
During the last few years, the antiviral effect of PCIs has also been observed against a number of viruses, including HIV. It has been described that HIV-1 replication could be affected by inhibiting CDKs (de la Fuente, et al. Current HIV Research, 2003, 1(2), 131-152; Y. K. Kim et al. Molecular and Cellular Biology, 2002; 22(13):4622-4637). In particular, CDK9 has been reported as being essential for HIV-1 replication. CDK9 forms together with cyclin T1 the human positive transcription elongation factor (PTEFb) regulating elongation phase of RNA polymerase II (RNA Pol II) dependent transcription (Price et al. Mol Cell Biol 2000, 20:2629-2634; Peterlin et al. Mol Cell 2006; 23:297-305). P-TEFb specifically activates transcription from the HIV-1 long-terminal repeat (LTR) promoter (Bieniasz et al. PNAS 1999; 96:7791-7796). After transcription initiation at the LTR, the newly transcribed transactivation responsive (TAR) RNA hairpin recruits the HIV-1 Tat protein, which binds to the cyclin T1 subunit of P-TEFb and recruits the kinase complex to phosphorylate Pol II (Bieniasz et al. PNAS 1999; 96:7791-7796, Zhang et al. J Biol Chem 2000; 275:34314-34319). HIV-1 Tat has recently been suggested to manipulate P-TEFb functional equilibrium by recruiting it from the large inactive complex thus increasing the active pool for efficient HIV-1 transcription (Barboric et al. Nucl Acids Res 2007; 35:2003-2012, Sedore Nucl Acids Res 2007; 35:4347-4358). The P-TEFb kinase activity is both essential and limiting for viral replication, and inhibition of P-TEFb by small molecules such as DRB 1 abrogates both viral transcription and replication (Flores et al. Proc. Natl. Acad. Sci. USA. 1999, 96(13):7208-13). The most potent P-TEFb inhibitor flavopiridol 2 effectively blocks HIV-1 Tat-transactivation and viral replication by inhibiting P-TEFb kinase activity at non-cytotoxic concentrations without affecting cellular transcription (Chao J Biol Chem 2000; 275:28345-28348, Chao J Biol Chem 2001; 276:31793-31799). RNAi-mediated gene silencing of PTEFb inhibits Tat-transactivation and HIV-1 replication in host cells with no effects on cell viability (Chiu J Virol 2004; 78:2517-2529). Furthermore, direct inhibition of CDK9 using a dominant negative form has been shown to potently inhibit HIV-1 replication without affecting RNA Pol II transcription and cell viability (Flores et al. Proc. Natl. Acad. Sci. USA. 1999, 96(13):7208-13, Salerno et al. Gene 2007; 405:65-78).
When pathogens infect a host, they must manipulate its signal-transduction pathways (the intracellular circuits that transmit extracellular signals from the cell surface to the nucleus and back) required for cellular function and survival (Coley et al. Exp Opin Biol Ther 2009). Preventing pathogens from hijacking such pathways via compounds exerting their action against certain cellular kinases that play key roles in cell signaling, generates a firewall against infection. The benefit of such an approach is that the target for the therapeutic intervention is directed primarily on the host-cell kinases, rather than on pathogen encoded targets. An important consequence of such an approach is that it minimizes the problem of drug resistance. Treatment with typical anti-infective drugs, which act directly on a pathogen encoded target, can be rendered ineffective due to generation of drug resistant variants (Thompson et al. JAMA 2012; 304(3):321-333, UK Coll Group J Inf Dis 2005). These drug resistant variants can occur rapidly, due to spontaneous mutations to the pathogen encoded target and poor proofreading mechanisms during the very short replication cycle of the pathogen. Mammalian host cellular enzymes are much less prone to mutations than microbial ones due to the fact that the mammalian host cell has a much more efficient proofreading mechanism and substantially longer replication cycle. Moreover, since targeting a host-cell kinase would be distal to a pathogen-encoded target this approach should therefore diminish the chances for developing pathogen resistance to such a drug.
Based on this rationale, we embarked on an initial screen for anti-HIV compounds chosen among a library of CDK9 inhibitors, PCT international application (published 30 Jun. 2011 as WO2011077171) “4-Phenylamino-Pyrimidine Derivatives having Protein Kinase Inhibitor Activity” (“PKI”) by Greff et al.
Unexpectedly, the results of the activity tests on such a library (which produced a number of initial hits) highlighted the lack of a direct correlation between CDK9 activity and the anticipated anti-HIV activity. In most cases there was no correlation whatsoever between CDK9 inhibition and anti-HIV activity, as compounds with potent CDK9-related IC50 exhibited poor anti-HIV profiles and vice versa.
This prompted us to explore a new approach: modifications on the original library compounds were proposed, synthetic routes were designed and reduced to practice by synthesizing the novel compounds, the compounds were then tested one by one, resulting in a novel class of molecules with the required properties, that are the subject of the present invention. As the CDK9 inhibitory activity and selectivity of these molecules is not predictive of the antiviral and/or antiproliferative activity they act by an as yet unknown mechanism.